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Distinct roles of MK2 and MK5 in cAMP/PKA- and stress/p38MAPK-induced heat shock protein 27 phosphorylation.

Shiryaev A, Dumitriu G, Moens U - J Mol Signal (2011)

Bottom Line: Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels.Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway.Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Tromsø, Faculty of Health Sciences, Department of Medical Biology, Host-Microbe Interaction Research Group, N-9037 Tromsø, Norway. Ugo.Moens@uit.no.

ABSTRACT

Background: Classical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three successive phosphorylation events resulting in the phosphorylation of a variety of substrates, including another class of protein kinases referred to as MAPK-activating protein kinases (MAPKAPKs). The MAPKAPKs MK2, MK3 and MK5 are closely related, but MK2 and MK3 are the major downstream targets of the p38MAPK pathway, while MK5 can be activated by the atypical MAPK ERK3 and ERK4, protein kinase A (PKA), and maybe p38MAPK. MK2, MK3, and MK5 can phosphorylate the common substrate small heat shock protein 27 (HSP27), a modification that regulates the role of HSP27 in actin polymerization. Both stress and cAMP elevating stimuli can cause F-actin remodeling, but whereas the in vivo role of p38MAPK-MK2 in stress-triggered HSP27 phosphorylation and actin reorganization is well established, it is not known whether MK2 is involved in cAMP/PKA-induced F-actin rearrangements. On the other hand, MK5 can phosphorylate HSP27 and cause cytoskeletal changes in a cAMP/PKA-dependent manner, but its role as HSP27 kinase in stress-induced F-actin remodeling is disputed. Therefore, we wanted to investigate the implication of MK2 and MK5 in stress- and PKA-induced HSP27 phosphorylation.

Results: Using HEK293 cells, we show that MK2, MK3, and MK5 are expressed in these cells, but MK3 protein levels are very moderate. Stress- and cAMP-elevating stimuli, as well as ectopic expression of active MKK6 plus p38MAPK or the catalytic subunit of PKA trigger HSP27 phosphorylation, and specific inhibitors of p38MAPK and PKA prevent this phosphorylation. Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels. Stimulation of the p38MAPK, but not the PKA pathway, caused activation of MK2.

Conclusion: Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway. Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization.

No MeSH data available.


Related in: MedlinePlus

Activation of the p38MAPK and PKA pathways can provoke phosphorylation of HSP27. (A) HEK293 cells were transfected with an expression vector for Flag-tagged HSP27 and 24 h after transfection left untreated (-) or exposed to forskolin (FSK; 10 μM for 30 min) or sodium arsenite (SA; 250 μM for 30 min). The phosphorylation levels of phosphoSer-78 HSP27 were monitored. Equal loading was verified by examining the total levels of HSP27 and actin in the samples. To ensure that the stimuli were active, phosphorylation of the PKA substrate CREB at Ser-133 and of the p38MAPK was tested. The double bands observed in the blots with (phospho)HSP27 antibodies represent Flag-tagged HSP27 (upper band) and endogenous HSP27 (lower band). (B) HEK293 cells transfected with the Flag-HSP27 expression plasmid and cells were either left untreated, exposed for 30 min with the PKA inhibitor H89 (10 μM) or the p38MAPK inhibitor SB203580 (10 μM) before forskolin or arsenite was added, or were treated with forskolin or arsenite as described in (A). Lane 1: untreated Flag-Hsp27 transfected HEK293 cells; lane 2: stimulated with forskolin; lane 3: stimulated with sodium arsenite; lane 4: exposed to H89; lane 5: pretreated with H89 and then stimulated with forskolin; lane 6: pretreated with H89 and then stimulated with arsenite; lane 7: exposed to SB203580; lane 8: pretreated with SB203580 and then stimulated with forskolin; lane 9: pretreated with SB203590 and then stimulated with arsenite. PhosphoSer78 HSP27 and phosphoSer82 HSP27 were examined. Membranes were stripped and the lysates were assayed for phosphorylation of CREB and p38MAPK. Lower panel shows the expression levels of endogenous and Flag-tagged HSP27. (C) Cells were cotransfected with expression vectors for Flag-tagged HSP27 and the catalytic subunit of PKA (Cα) or p38MAPK and a constitutive active mutant of its upstream activator MKK6 (MKK). PhosphoSer78 HSP27 protein levels were examined by western blotting. M is the protein molecular mass (in kD) marker. RDU (relative densitometry units) indicates the increase in HSP27 phosphorylation and was calculated as follows. The densitometry values obtained for the signals of phosphorylated HSP27 were determined and corrected for the values obtained for actin (in A) or for total HSP27 (in B). This ratio obtained for untreated cells was arbitrarily set as 1.0 and the ratios obtained for stimulated cells were related to this. Values were calculated separately for flag-tagged HSP27 and endogenous HSP27.
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Figure 3: Activation of the p38MAPK and PKA pathways can provoke phosphorylation of HSP27. (A) HEK293 cells were transfected with an expression vector for Flag-tagged HSP27 and 24 h after transfection left untreated (-) or exposed to forskolin (FSK; 10 μM for 30 min) or sodium arsenite (SA; 250 μM for 30 min). The phosphorylation levels of phosphoSer-78 HSP27 were monitored. Equal loading was verified by examining the total levels of HSP27 and actin in the samples. To ensure that the stimuli were active, phosphorylation of the PKA substrate CREB at Ser-133 and of the p38MAPK was tested. The double bands observed in the blots with (phospho)HSP27 antibodies represent Flag-tagged HSP27 (upper band) and endogenous HSP27 (lower band). (B) HEK293 cells transfected with the Flag-HSP27 expression plasmid and cells were either left untreated, exposed for 30 min with the PKA inhibitor H89 (10 μM) or the p38MAPK inhibitor SB203580 (10 μM) before forskolin or arsenite was added, or were treated with forskolin or arsenite as described in (A). Lane 1: untreated Flag-Hsp27 transfected HEK293 cells; lane 2: stimulated with forskolin; lane 3: stimulated with sodium arsenite; lane 4: exposed to H89; lane 5: pretreated with H89 and then stimulated with forskolin; lane 6: pretreated with H89 and then stimulated with arsenite; lane 7: exposed to SB203580; lane 8: pretreated with SB203580 and then stimulated with forskolin; lane 9: pretreated with SB203590 and then stimulated with arsenite. PhosphoSer78 HSP27 and phosphoSer82 HSP27 were examined. Membranes were stripped and the lysates were assayed for phosphorylation of CREB and p38MAPK. Lower panel shows the expression levels of endogenous and Flag-tagged HSP27. (C) Cells were cotransfected with expression vectors for Flag-tagged HSP27 and the catalytic subunit of PKA (Cα) or p38MAPK and a constitutive active mutant of its upstream activator MKK6 (MKK). PhosphoSer78 HSP27 protein levels were examined by western blotting. M is the protein molecular mass (in kD) marker. RDU (relative densitometry units) indicates the increase in HSP27 phosphorylation and was calculated as follows. The densitometry values obtained for the signals of phosphorylated HSP27 were determined and corrected for the values obtained for actin (in A) or for total HSP27 (in B). This ratio obtained for untreated cells was arbitrarily set as 1.0 and the ratios obtained for stimulated cells were related to this. Values were calculated separately for flag-tagged HSP27 and endogenous HSP27.

Mentions: To test whether activation of the PKA pathway results in phosphorylation of HSP27, Flag-tagged HSP27 transfected HEK293 cells were treated with the cAMP elevating agent forskolin and phosphorylation of HSP27 at Ser-78 and Ser-82 was monitored by western blot using phosphoSer-78 and phosphoSer-82 specific antibodies. Forskolin treatment resulted in >2-fold increase in HSP27 phosphorylation at Ser-78 and Ser-82 (Figure 3A and 3B). Activation of the p38MAPK pathway by sodium arsenite triggered HSP27 phosphorylation at both sites (Figure 3A and 3B). Forskolin (respectively arsenite) treatment enhanced phosphorylation of the PKA substrate CREB (respectively phosphorylation of p38MAPK), indicating that the stimuli were functional (Figure 3A). In agreement with previous studies, arsenite also caused CREB phosphorylation, probably through MSK1 [4,8]. Pretreatment of the cells with the PKA specific inhibitor H89 reduced forskolin-induced HSP27 phosphorylation, indicating the involvement of PKA (Figure 3B). The specific p38MAPK inhibitor SB203580 almost completely abrogated arsenite-induced HSP27 phosphorylation (Figure 3B). To provide further experimental proof that PKA can mediate phosphorylation of HSP27, we cotransfected cells with expression plasmids for the catalytic subunit of PKA (Cα) and Flag-tagged HSP27. Increased phosphorylation of endogenous and Flag-tagged HSP27 was observed in cells ectopically expressing PKA-Cα compared to cells transfected with empty vector (compare lanes 1 and 2 in Figure 3C).


Distinct roles of MK2 and MK5 in cAMP/PKA- and stress/p38MAPK-induced heat shock protein 27 phosphorylation.

Shiryaev A, Dumitriu G, Moens U - J Mol Signal (2011)

Activation of the p38MAPK and PKA pathways can provoke phosphorylation of HSP27. (A) HEK293 cells were transfected with an expression vector for Flag-tagged HSP27 and 24 h after transfection left untreated (-) or exposed to forskolin (FSK; 10 μM for 30 min) or sodium arsenite (SA; 250 μM for 30 min). The phosphorylation levels of phosphoSer-78 HSP27 were monitored. Equal loading was verified by examining the total levels of HSP27 and actin in the samples. To ensure that the stimuli were active, phosphorylation of the PKA substrate CREB at Ser-133 and of the p38MAPK was tested. The double bands observed in the blots with (phospho)HSP27 antibodies represent Flag-tagged HSP27 (upper band) and endogenous HSP27 (lower band). (B) HEK293 cells transfected with the Flag-HSP27 expression plasmid and cells were either left untreated, exposed for 30 min with the PKA inhibitor H89 (10 μM) or the p38MAPK inhibitor SB203580 (10 μM) before forskolin or arsenite was added, or were treated with forskolin or arsenite as described in (A). Lane 1: untreated Flag-Hsp27 transfected HEK293 cells; lane 2: stimulated with forskolin; lane 3: stimulated with sodium arsenite; lane 4: exposed to H89; lane 5: pretreated with H89 and then stimulated with forskolin; lane 6: pretreated with H89 and then stimulated with arsenite; lane 7: exposed to SB203580; lane 8: pretreated with SB203580 and then stimulated with forskolin; lane 9: pretreated with SB203590 and then stimulated with arsenite. PhosphoSer78 HSP27 and phosphoSer82 HSP27 were examined. Membranes were stripped and the lysates were assayed for phosphorylation of CREB and p38MAPK. Lower panel shows the expression levels of endogenous and Flag-tagged HSP27. (C) Cells were cotransfected with expression vectors for Flag-tagged HSP27 and the catalytic subunit of PKA (Cα) or p38MAPK and a constitutive active mutant of its upstream activator MKK6 (MKK). PhosphoSer78 HSP27 protein levels were examined by western blotting. M is the protein molecular mass (in kD) marker. RDU (relative densitometry units) indicates the increase in HSP27 phosphorylation and was calculated as follows. The densitometry values obtained for the signals of phosphorylated HSP27 were determined and corrected for the values obtained for actin (in A) or for total HSP27 (in B). This ratio obtained for untreated cells was arbitrarily set as 1.0 and the ratios obtained for stimulated cells were related to this. Values were calculated separately for flag-tagged HSP27 and endogenous HSP27.
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Related In: Results  -  Collection

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Figure 3: Activation of the p38MAPK and PKA pathways can provoke phosphorylation of HSP27. (A) HEK293 cells were transfected with an expression vector for Flag-tagged HSP27 and 24 h after transfection left untreated (-) or exposed to forskolin (FSK; 10 μM for 30 min) or sodium arsenite (SA; 250 μM for 30 min). The phosphorylation levels of phosphoSer-78 HSP27 were monitored. Equal loading was verified by examining the total levels of HSP27 and actin in the samples. To ensure that the stimuli were active, phosphorylation of the PKA substrate CREB at Ser-133 and of the p38MAPK was tested. The double bands observed in the blots with (phospho)HSP27 antibodies represent Flag-tagged HSP27 (upper band) and endogenous HSP27 (lower band). (B) HEK293 cells transfected with the Flag-HSP27 expression plasmid and cells were either left untreated, exposed for 30 min with the PKA inhibitor H89 (10 μM) or the p38MAPK inhibitor SB203580 (10 μM) before forskolin or arsenite was added, or were treated with forskolin or arsenite as described in (A). Lane 1: untreated Flag-Hsp27 transfected HEK293 cells; lane 2: stimulated with forskolin; lane 3: stimulated with sodium arsenite; lane 4: exposed to H89; lane 5: pretreated with H89 and then stimulated with forskolin; lane 6: pretreated with H89 and then stimulated with arsenite; lane 7: exposed to SB203580; lane 8: pretreated with SB203580 and then stimulated with forskolin; lane 9: pretreated with SB203590 and then stimulated with arsenite. PhosphoSer78 HSP27 and phosphoSer82 HSP27 were examined. Membranes were stripped and the lysates were assayed for phosphorylation of CREB and p38MAPK. Lower panel shows the expression levels of endogenous and Flag-tagged HSP27. (C) Cells were cotransfected with expression vectors for Flag-tagged HSP27 and the catalytic subunit of PKA (Cα) or p38MAPK and a constitutive active mutant of its upstream activator MKK6 (MKK). PhosphoSer78 HSP27 protein levels were examined by western blotting. M is the protein molecular mass (in kD) marker. RDU (relative densitometry units) indicates the increase in HSP27 phosphorylation and was calculated as follows. The densitometry values obtained for the signals of phosphorylated HSP27 were determined and corrected for the values obtained for actin (in A) or for total HSP27 (in B). This ratio obtained for untreated cells was arbitrarily set as 1.0 and the ratios obtained for stimulated cells were related to this. Values were calculated separately for flag-tagged HSP27 and endogenous HSP27.
Mentions: To test whether activation of the PKA pathway results in phosphorylation of HSP27, Flag-tagged HSP27 transfected HEK293 cells were treated with the cAMP elevating agent forskolin and phosphorylation of HSP27 at Ser-78 and Ser-82 was monitored by western blot using phosphoSer-78 and phosphoSer-82 specific antibodies. Forskolin treatment resulted in >2-fold increase in HSP27 phosphorylation at Ser-78 and Ser-82 (Figure 3A and 3B). Activation of the p38MAPK pathway by sodium arsenite triggered HSP27 phosphorylation at both sites (Figure 3A and 3B). Forskolin (respectively arsenite) treatment enhanced phosphorylation of the PKA substrate CREB (respectively phosphorylation of p38MAPK), indicating that the stimuli were functional (Figure 3A). In agreement with previous studies, arsenite also caused CREB phosphorylation, probably through MSK1 [4,8]. Pretreatment of the cells with the PKA specific inhibitor H89 reduced forskolin-induced HSP27 phosphorylation, indicating the involvement of PKA (Figure 3B). The specific p38MAPK inhibitor SB203580 almost completely abrogated arsenite-induced HSP27 phosphorylation (Figure 3B). To provide further experimental proof that PKA can mediate phosphorylation of HSP27, we cotransfected cells with expression plasmids for the catalytic subunit of PKA (Cα) and Flag-tagged HSP27. Increased phosphorylation of endogenous and Flag-tagged HSP27 was observed in cells ectopically expressing PKA-Cα compared to cells transfected with empty vector (compare lanes 1 and 2 in Figure 3C).

Bottom Line: Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels.Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway.Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Tromsø, Faculty of Health Sciences, Department of Medical Biology, Host-Microbe Interaction Research Group, N-9037 Tromsø, Norway. Ugo.Moens@uit.no.

ABSTRACT

Background: Classical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three successive phosphorylation events resulting in the phosphorylation of a variety of substrates, including another class of protein kinases referred to as MAPK-activating protein kinases (MAPKAPKs). The MAPKAPKs MK2, MK3 and MK5 are closely related, but MK2 and MK3 are the major downstream targets of the p38MAPK pathway, while MK5 can be activated by the atypical MAPK ERK3 and ERK4, protein kinase A (PKA), and maybe p38MAPK. MK2, MK3, and MK5 can phosphorylate the common substrate small heat shock protein 27 (HSP27), a modification that regulates the role of HSP27 in actin polymerization. Both stress and cAMP elevating stimuli can cause F-actin remodeling, but whereas the in vivo role of p38MAPK-MK2 in stress-triggered HSP27 phosphorylation and actin reorganization is well established, it is not known whether MK2 is involved in cAMP/PKA-induced F-actin rearrangements. On the other hand, MK5 can phosphorylate HSP27 and cause cytoskeletal changes in a cAMP/PKA-dependent manner, but its role as HSP27 kinase in stress-induced F-actin remodeling is disputed. Therefore, we wanted to investigate the implication of MK2 and MK5 in stress- and PKA-induced HSP27 phosphorylation.

Results: Using HEK293 cells, we show that MK2, MK3, and MK5 are expressed in these cells, but MK3 protein levels are very moderate. Stress- and cAMP-elevating stimuli, as well as ectopic expression of active MKK6 plus p38MAPK or the catalytic subunit of PKA trigger HSP27 phosphorylation, and specific inhibitors of p38MAPK and PKA prevent this phosphorylation. Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels. Stimulation of the p38MAPK, but not the PKA pathway, caused activation of MK2.

Conclusion: Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway. Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization.

No MeSH data available.


Related in: MedlinePlus